Development of a functional and efficient nervous system requires the orchestrated specification, migration and differentiation of glia. During gliogenesis in both the central and peripheral nervous systems (CNS and PNS, respectively), large populations of glia are specified that must migrate and differentiate into not only functional glial cells, but also coordinate their development so that they occupy discrete, non-overlapping territories with neighboring glia. This phenomenon of glial spacing, or tiling, can occur between glia found in the CNS, PNS, or between glial cells where one cell resides in the CNS and the other resides in the PNS. Although we know that these tiling events occur, we don?t know the molecular nature of these interactions or whether they are used by all tiling glia. Additionally, how local glial-glial interactions play a role in global glial tiling is unknown. In this proposal, using zebrafish as a model organism, we will investigate the cellular and molecular mechanisms that mediate pre-myelinating glial tiling at motor exit point (MEP) transition zones (TZ) and in the developing spinal cord. In Aim 1 of this project, we will use a combination of expression analysis, multi-color, in vivo, time-lapse imaging, electron microscopy, pharmacological and genetic manipulation (CRISPR) of newly identified mediators to elucidate the cellular and molecular mechanisms that govern myelinating glial tiling at MEP TZs. In Aim 2, we will determine if the same mechanisms we characterize in Aim 1 also mediate tiling between oligodendrocyte progenitor cells (OPC) in the developing spinal cord. To better understand, diagnose and treat the many degenerative disorders of the CNS and PNS, we need to comprehend the cellular and molecular mechanisms that mediate glial-glial interactions and tiling. Zebrafish provide a unique opportunity to directly observe and manipulate cell populations to gain insight into how glial cells interact under normal physiological conditions, and if those interactions that ultimately result in glial tiling are perturbed in disease.